In this proposal we focus on exploring how cells achieve specificity in the early steps of protein import into mitochondria. As model system we use the mitochondrial isozyme of aspartate aminotransferase (mAAT) which resides in the matrix of the organelle, and its highly homologous cytosolic counterpart (cAAT). Both proteins are encoded in the nuclear genome and synthesized in the cytoplasm. mAAT originates as a precursor form (pmAAT) containing an N-terminal extension or presequence peptide. This presequence peptide is necessary, but not sufficient, to target the protein to the mitochondrial membrane for subsequent translocation into the organelle. After import to the matrix, the presence is proteolytically removed. Import also requires a partially unfolded conformation of the passenger protein. Apparently, various proteins in the cytosol, acting as molecular chaperones, slow down the folding of mitochondrial precursors, maintaining them in an import-competent conformation until they can engage the import machinery of the mitochondrial membrane. This cytosolic component(s) discriminate between the two enzymes showing strict selectivity for mAAT. In addition, folding intermediates of mAAT (but not cAAT) bind avidly to phospholipid vesicles or detergent micelles. At present, very little is known about the conformation of the passenger protein complexed to the cytosolic factors or the mitochondrial membrane surface, the series of sequential events involved in its transfer to the components of the outer membrane responsible for the recognition and initiation of the translocation process and, the structural features of the mitochondrial protein that determine its selective sorting and import. With our ability to express in and isolate from E. coli the wild type form, and a great variety of different engineered species of an authentic mitochondrial precursor protein, pmAAT, as well as cAAT, we are in a unique position to address questions related to the early events of the translocation of proteins into mitochondria. The main goals of this proposal are to investigate 1) how much structure there is in the polypeptide chain that is translocation competent and possible mechanisms of achieving or stabilizing this particular conformational state, 2) the elements in the structure of the translocated protein responsible for its selective recognition and binding to the mitochondria, and 3) the structural requirements of the border region between the presequence and the N-terminus of the mature part of the protein for proper processing of the precursor.